Insulin ELISA AFM STUDY MATERIAL

2. Determination of blood Insulin
concentration by enzyme
linked immunosorbent assay
(ELISA)
Prepared and presented
By Dr. Mohamed Agha

3. Cases
 CASE I:
A 50-year-old female presents to your clinic with
complaints of excessive thirst, fluid intake, and
urination. She denies any urinary tract infection
symptoms. She reports no medical problems, but has
not seen a doctor in many years. On examination she
is an obese female in no acute distress. Her physical
exam is otherwise normal. The urine analysis revealed
high glucose level, and a serum random blood sugar
level was 320 mg/dl.
What is the most likely diagnosis?
A case of type II DM (might be insulin resistance state).

4.  CASE II
 An 82-year-old white woman presented to her
primary care physician with history of episodic
confusion and somnolence. The episodes occurred
typically in the morning, just after waking. They
lasted minutes and were relieved when she ate her
breakfast or had juice. As she was waiting for
check-out, she developed confusion, a capillary
blood glucose test was performed, and she was
noted to have a plasma glucose level of 28 mg/dl.
She was given juice and her symptoms resolved
after a few moments.
 What is the most likely diagnosis?
A case of insulinomas (islet cell tumors of the
pancreas).

5. Insulin
 Human insulin is a peptide hormone
composed of 51 amino acids.
 It is produced in the islets of
Langerhans in the pancreas.
 It is a hormone central to regulating
carbohydrate and fat metabolism in
the body.

6. Diseases associated with
insulin disturbances
 Elevated insulin levels are seen with:
1. Insulinomas
2. Insulin resistance, such as in obesity,
Diabetes (Type 2) and metabolic
syndrome.
 Decreased insulin levels are seen with:
1. Diabetes (Type 1)
2. Pancreatic diseases such as chronic
pancreatitis (including cystic fibrosis) and

7. Enzyme Linked Immunosorbent
Assay
(ELISA)
 Definition:
This is a sensitive and specific enzyme-
based immunoassay that is used for
quantification of a substance even of
picograms (10-12g) range (ex. hormones,
drugs, pathogens).
 The name suggests three
components:
1. Antibody.
2. Solid phase (sorbent).
3. Enzymatic amplification.

8. Principle:
 Antigen:
a high molecular weight (>5000) substance
recognized by the immune system of an
organism as foreign.
The organism reacts to antigen by producing
a specific antibody.
 Hapten:
a low molecular weight (<5000) substance
can´t stimulate the production of antibodies.
It must be carried by a specific carrier to
stimulate the production of a specific
antibody.

9.  Antibody (immunoglobulin):
a large Y-shaped protein
used by the immune system to identify
and neutralize foreign objects such as
bacteria and viruses.
recognizes a unique part of the foreign
target, termed an antigen.
 ELISA:
is based on an antigen-antibody
(immunological) reaction

10. Equipment:
 Microtiter plate
 Microplate sealing tape
 Automatic pipettes

11.  ELISA washer
 ELISA reader

12. Types of ELISA:
Indirect ELISA
Sandwich ELISA
Competitive ELISA

13. Indirect ELISA
 Used to measure
antibody levels.

 The antibody reacts with
specific antigen attached to
the solid phase.

 Enzyme activity
measured is proportional
to the amount of specific
antibody in the original
serum.

 Color produced is directly
proportional to antibody

14. Indirect ELISA

15. Sandwich ELISA
 The double antibody
method.
 Used to measure antigen
levels.
 The unknown antigen reacts
with specific antibody
attached to the solid phase.
 Enzyme activity measured
is proportional to the
amount of specific antigen in
the original solution.
 Color produced is directly
proportional to antigen
amount.

16. Sandwich ELISA

17. Competitive ELISA
 Enzyme- labeled antigen
and the unknown amount of
the antigen in the sample
react with a specific
antibody attached to a solid
phase.
 Enzyme activity measured
is proportional to the
proportion of labelled
antigen in mixture of
labelled and unlabelled
antigen.
 Color produced is inversely

18. Principle of the insulin ELISA
 Based on the sandwich principle.
 The microtiter wells are coated with a monoclonal
antibody directed towards Insulin molecule.
 Samples and standards are pipetted into these wells,
along with the enzyme conjugate (monoclonal
antibody against human insulin, which has
horseradish peroxidase (HRP) enzyme covalently
linked to it).
 The amount of bound HRP complex is proportional to
the concentration of Insulin in the sample.

19.  After washing to remove unbound
materials, a substrate for HRP is
added.
 The substrate is converted to a
colored compound by the action of
HRP.
 The intensity of colour developed is
proportional to the concentration of
Insulin in the patient sample.

20. Assay Procedure